Activity of Pseudomonas Type III Toxins

描述（由申请方提供）：铜绿假单胞菌是一种 一种机会致病菌，表达多种毒力决定因子。 我们的工作集中在一种胞外细菌产物的贡献上 致病机制，外切酶S。外切酶S是一个家族成员， ADP-核糖基转移酶。外切酶S的产生与 铜绿假单胞菌从上皮细胞扩散或播散能力 感染个体的血液中的定植位点，导致 致命性败血症的发展。我们最初的中毒机制模型 对于ExoS的评估过于简单，并且基于ExoS将展示A：B的概念 结构然而，我们随后发现，ExoS被运送到了 通过III型中毒机制在真核细胞的胞质溶胶中形成。这些 观察结果开辟了新的研究领域， 假单胞菌III型系统的序列分析，表征主要的 由III型装置分泌的细胞外蛋白，并发现两个 新型毒素ExoU和ExoY这项新提案的长期目标是 确定ExoU的作用机制并开始表达， ExoY的生化研究。ExoU表达导致急性 培养细胞的细胞毒性反应和体内肺损伤。ExoU没有 已知基序、酶活性或与数据中其他蛋白质的同源性 很可能是一种新型毒素结构功能分析表明 ExoU的细胞毒性应答在至少两个结构域中编码， 在哺乳动物细胞中起反式作用。新数据，在此 应用，证明ExoU对酵母有毒。我们将使用酵母作为 模型遗传和生化系统，以确定ExoU毒性的目标。 ExoY具有腺苷酸环化酶活性，与腺苷酸环化酶活性相关。 炭疽杆菌和百日咳杆菌环化酶毒素。虽然 ExoY的活性是已知的，ExoY表达和ExoY表达之间的关联是已知的。 活性相对于铜绿假单胞菌的发病机制尚未被检查。 了解ExoU和ExoY的作用机制将有助于设计 替代治疗的方法。

DESCRIPTION (provided by the applicant): Pseudomonas aeruginosa is an opportunistic pathogen that expresses a wide variety of virulence determinants. Our work has focused on the contribution of one extracellular bacterial product to pathogenesis, exoenzyme S. Exoenzyme S is a member of the family of ADP-ribosyltransferase enzymes. Production of exoenzyme S is correlated with the ability of P. aeruginosa to spread or disseminate from epithelial colonization sites to the bloodstream of infected individuals, resulting in the development of a fatal sepsis. Our initial models of the intoxication mechanism for ExoS were simplistic and based on the notion that ExoS would exhibit an A:B structure. We subsequently showed, however, that ExoS was delivered into the cytosol of eukaryotic cells by a type III mechanism of intoxication. These observations opened new areas of investigation resulting in the cloning and sequence analysis of the Pseudomonas type III system, characterizing the major extracellular proteins secreted by the type III apparatus, and discovering two new toxins, ExoU and ExoY. The long-term goals of this new proposal are to determine the mechanism of action of ExoU and to begin expression and biochemical studies on ExoY. ExoU expression is responsible for the acute cytotoxic response in cultured cells and lung injury in vivo. ExoU possesses no known motifs, enzymatic activity, or homology to other proteins in the data base and likely represents a novel toxin. Structure-function analysis indicates that the cytotoxic response of ExoU is encoded in at least two domains that can function in trans within mammalian cells. New data, presented in this application, demonstrate that ExoU is toxic to yeast. We will use yeast as a model genetic and biochemical system to identify the target of ExoU toxicity. ExoY possesses adenylate cyclase activity and is related to the adenylate cyclase toxins of Bacillus anthracis and Bordetella pertussis. Although the activity of ExoY is known, the association of ExoY expression and the ExoY activity have not been examined relative to the pathogenesis of P. aeruginosa. Understanding the mechanisms of action of ExoU and ExoY will aid in the design of alternative treatments.